Stably transfected rodent fibroblast cell lines expressing human GABA-A -receptors

ABSTRACT

The present invention relates to stably co-transfected eukaryotic cell lines capable of expressing a recombinant GABA A  receptor, particularly a recombinant human GABA A  receptor, which comprises at least one alpha, one beta and one gamma subunit; and to the use of the cell line and/or membrane preparation in selecting compounds and designing medicaments which interact with the respective human recombinant GABA A  receptor.

This invention concerns a cell line, and in particular relates to a stable cell line capable of expressing human or animal GABA_(A) receptors. The invention further concerns the cloning of novel cDNA sequences encoding particular subunits of the human GABA_(A) receptor. In addition, the invention relates to the use of the cell line in a screening technique for the design and development of subtype-specific medicaments.

Gamma-amino butyric acid (GABA) is a major inhibitory neurotransmitter in the central nervous system. It mediates fast synaptic inhibition by opening the chloride channel intrinsic to the GABA_(A) receptor. This receptor comprises a multimeric protein of molecular size 230-270 kDa with specific binding sites for a variety of drugs including benzodiazepines, barbiturates and β-carbolines, in addition to sites for the agonist ligand GABA (for reviews see Stephenson, Biochem. J., 1988, 249, 21; Olsen and Tobin, Faseb J., 1990, 4, 1469; and Sieghart, Trends in Pharmacol. Sci., 1989, 10, 407).

Molecular biological studies demonstrate that the receptor is composed of several distinct types of subunit, which are divided into four classes (α, β, γ, and δ) based on their sequence similarities. To date, six types of α (Schofield et al., Nature (London), 1987, 328, 221; Levitan et al., Nature (London), 1988, 335, 76; Ymer et al., EMBO J., 1989, 8, 1665; Pritchett & Seeberg, J. Neurochem., 1990, 54, 802; Luddens et al., Nature (London), 1990, 346, 648; and Khrestchatisky et al., Neuron, 1989, 3, 745), three types of β (Ymer et al., EMBO J., 1989, 8, 1665), two types of γ (Ymer et al., EMBO J., 1990, 9, 3261; and Shivers et al., Neuron, 1989, 3, 327) and one δ subunit (Shivers et al., Neuron, 1989, 3, 327) have been identified.

The differential distribution of many of the subunits has been characterised by in situ hybridisation (Sequier et al., Proc. Natl. Acad. Sci. USA, 1988, 85, 7815; Malherbe et al., J. Neurosci., 1990, 10, 2330; and Shivers et al., Neuron, 1989, 3, 327) and this has permitted it to be speculated which subunits, by their co-localisation, could theoretically exist in the same receptor complex.

Various combinations of subunits have been co-transfected into cells to identify synthetic combinations of subunits whose pharmacology parallels that of bona fide GABA_(A) receptors in vivo (Pritchett et al., Science, 1989, 245, 1389; Malherbe et al., J. Neurosci., 1990, 10, 2330; Pritchett and Seeberg, J. Neurochem., 1990, 54, 1802; and Luddens et al., Nature (London), 1990, 346, 648). This approach has revealed that, in addition to an α and β subunit, either γ₁ or γ₂ (Pritchett et al., Nature (London), 1989, 338, 582; Ymer et al., EMBO J., 1990, 9, 3261; and Malherbe et al., J. Neurosci., 1990, 10, 2330) or γ₃ (Herb et al., Proc. Natl. Acad. Sci. USA, 1992, 89, 1433; Knoflach et al., FEBS Lett., 1991, 293, 191; and Wilson-Shaw et al., FEBS Lett., 1991, 284, 211) is also generally required to confer benzodiazepine sensitivity, and that the benzodiazepine pharmacology of the expressed receptor is largely dependent on the identity of the α and γ subunits present. Receptors containing a δ subunit (i.e. αβδ) do not appear to bind benzodiazepines (Shivers et al., Neuron, 1989, 3, 327). Combinations of subunits have been identified which exhibit the pharmacological profile of a BZ₁ type receptor (α₁ β₁ γ₂) and a BZ₂ type receptor (α₂ β₁ γ₂ or α₃ β₁ γ₂, Pritchett et al., Nature (London), 1989, 338, 582), as well as two GABA_(A) receptors with a novel pharmacology, α₅ β₂ γ₂ (Pritchett and Seeberg, J. Neurochem., 1990, 54, 1802) and α₆ β₂ γ₂ (Luddens et al., Nature (London), 1990, 346, 648). Although the pharmacology of these expressed receptors appears similar to that of those identified in brain tissue by radioligand binding, it has nonetheless not been shown that these receptor subunit combinations exist in vivo.

FIG. 1 shows the DNA plasmid expression vector pMSGneo. The designation "R" represents the nucleotide sequence of a given alpha, beta or gamma subunit of the GABA_(A) receptor, fused downstream of the inducible MMTV-LTR promoter fragment.

FIG. 2 shows the nucleotide sequence (SEQ ID NO:7) and deduced amino acid sequence (SED ID NO:8) encoding the human GABA_(A) receptor α₂ subunit.

FIG. 3 shows the nucleotide sequence (SEQ ID NO:9) and deduced amino acid sequence (SEQ ID NO:10) encoding the human GABA_(A) receptor α₃ subunit.

FIG. 4 shows the nucleotide sequence (SEQ ID NO:1) and deduced amino acid sequence (SEQ ID NO:2) encoding the human GABA_(A) receptor α₅ subunit.

FIG. 5 shows the nucleotide sequence (SEQ ID NO:3) and deduced amino acid sequence (SEQ ID NO:4) encoding the human GABA_(A) receptor α₆ subunit.

FIG. 6 shows the nucleotide sequence (SEQ ID NO:5) and deduced amino acid sequence (SEQ ID NO:6) encoding the human GABA_(A) receptor β₂ subunit.

The present invention is concerned with the production of permanently transfected cells containing the GABA_(A) receptor, which will be useful for screening for drugs which act on this receptor. The GABA_(A) receptor has previously been expressed in Xenopus oocytes (Sigel et al., Neuron, 1990, 5, 703-711) and in transiently transfected mammalian cells (Pritchett et al., Science, 1989, 245, 1389-1392). However, both of those systems involve transient expression and are unsuitable for screening purposes.

We have now achieved the stable expression of the receptor.

Accordingly, the present invention provides a stably co-transfected eukaryotic cell line capable of expressing a GABA_(A) receptor, which receptor comprises at least one alpha, one beta and one gamma subunit.

This has been achieved by co-transfecting cells with three expressien vectors, each harbouring cDNAs encoding for an α, β or γ GABA_(A) receptor subunit. In a further aspect, therefore, the present invention provides a process for the preparation of a eukarvotic cell line capable of expressing a GABA_(A) receptor, which comprises stably co-transfecting a eukaryotic host cell with at least three expression vectors, one such vector harbouring the cDNA sequence encoding for an alpha, another such vector harbouring the cDNA sequence encoding for a beta, and a third such vector harbouring the cDNA sequence encoding for a gamma GABA_(A) receptor subunit. The stable cell-line which is established expresses an αβγ GABA_(A) receptor. Each receptor thereby expressed, comprising a unique combination of α, β and γ subunits, will be referred to hereinafter as a GABA_(A) receptor "subunit combination". Pharmacological and electrophysiological data confirm that the recombinant αβγ receptor expressed by the cells of the present invention has the properties expected of a native GABA_(A) receptor.

Expression of the GABA_(A) receptor may be accomplished by a variety of different promoter-expression systems in a variety of different host cells. The eukaryotic host cells suitably include yeast, insect and mammalian cells. Preferably the eukaryotic cells which can provide the host for the expression of the receptor are mammalian cells. Suitable host cells include rodent fibroblast lines, for example mouse Ltk⁻, Chinese hamster ovary (CHO) and baby hamster kidney (BHK); HeLa; and HEK293 cells. It is necessary to incorporate at least one α, one β and one γ subunit into the cell line in order to produce the required receptor. Within this limitation, the choice of receptor subunit combination is made according to the type of activity or selectivity which is being screened for. For example, benzodiazepines (designated BZ) represent one class of drugs which act upon the GABA_(A) receptor. The presence of an α₁ subunit is specific for a class of benzodiazepines having the pharmacology designated BZ₁ ; whereas α₂ to α₅ define different pharmacological profiles, broadly designated as BZ₂. The type of β subunit is not critical in defining the class of benzodiazepine, although a β subunit is required. The γ subunit is also important in defining BZ selectivity. It is likely that differentiation between α subunit selectivity is conferred by the identity of the particular γ subunit present.

In order to employ this invention most effectively for screening purposes, it is preferable to build up a library of cell lines, each with a different combination of subunits. Typically a library of 5 or 6 cell line types is convenient for this purpose. Preferred subunit combinations include: α₁ β₁ γ₂ ; α₁ β₂ γ₂ ; α₂ β₁ γ₁ ; α₂ β₁ γ₂ ; α₂ β₁ γ₃ ; α₃ β₁ γ₂ ; α₃ β₁ γ₃ ; α₄ β₁ γ₂ ; α₅ β₁ γ₂ ; and α₆ β₁ γ₂ ; especially α₁ β₁ γ_(2L).

In a particular embodiment, the present invention provides a stably co-transfected eukaryotic cell line capable of expressing a human GABA_(A) receptor comprising the α₁ β₃ γ₂ subunit combination.

In a further embodiment, the present invention provides a stably co-transfected eukaryotic cell line capable of expressing a human GABA_(A) receptor comprising the α₂ β₃ γ₂ subunit combination.

In a still further embodiment, the present invention provides a stably co-transfected eukaryotic cell line capable of expressing a human GABA_(A) receptor comprising the α₅ β₃ γ₂ subunit combination.

In yet further embodiments, the present invention provides stably co-transfected eukaryotic cell lines capable of expressing human GABA_(A) receptors comprising the α₁ β₁ γ_(2S), α₁ β₂ γ₂, α₃ β₃ γ₂ and α₆ β₃ γ₂ subunit combinations.

The DNAs for the receptor subunits can be obtained from known sources, and are generally obtained as specific nucleotide sequences harboured by a standard cloning vector such as those described, for example, by Maniatis et al. in Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, New York, 2nd edition, 1989. Preferably the cDNA sequences are derived from the human gene. However, for screening purposes, cDNAs from other species are also suitable, such as bovine or rat DNA. Known sources of GABA_(A) receptor subunit cDNAs are as follows:

    ______________________________________      α.sub.1 bovine                     Schofield et al., Nature, 1987, 328,     β.sub.1 bovine                    221-227.      α.sub.1 human                     Schofield et al., FEBS Lett., 1989, 244,     β.sub.1 human                    361-364.     α.sub.2 rat                    Khrestchatisky et al., J. Neurochem.,                    1991, 56, 1717.      α.sub.2 bovine                     Levitan et al., Nature, 1988, 335,     α.sub.3 bovine                    76-79.     α.sub.4 rat                    Wisden et al., FEBS Lett., 1991, 289, 227.     α.sub.4 bovine                    Ymer et al., FEBS Lett., 1989, 258,                    119-122.     α.sub.5 rat                    Pritchett and Seeburg,                    J. Neurochem., 1990, 54, 1802-1804.      α.sub.6 rat                     Luddens et al., Nature, 1990, 346,     α.sub.6 bovine                    648-651.     β.sub.2 bovine     β.sub.2 rat     β.sub.3 bovine                    Ymer et al., EMBO J., 1989, 8, 1665-1670.     β.sub.3 rat     β.sub.3 human                    Wagstaff et al., Am. J. Hum. Genet., 1991,                    49, 330.     γ.sub.1 human     γ.sub.1 rat                    Ymer et al., EMBO J., 1990, 9, 3261-3267.     γ.sub.1 bovine     γ.sub.2 human                    Pritchett et al., Nature, 1989, 338,                    582-585.     γ.sub.2 bovine                    Whiting et al., Proc. Natl. Acad.                    Sci. USA, 1990, 57, 9966-9970.     γ.sub.3 rat                    Herb et al., Proc. Natl. Acad. Sci. USA,                    1992, 89, 1433; and                    Knoflach et al., FEBS Lett., 1991, 293,                    191.     γ.sub.3 mouse                    Wilson-Shaw et al., FEBS Lett., 1991, 284,                    211.     δ rat    Shivers et al., Neuron, 1989, 3, 327.     ______________________________________

Certain cDNA sequences encoding various subunits of the human GABA_(A) receptor have hitherto been unavailable. These include in particular the sequences encoding the α₂, α₃, α₅, α₆ and β₂ subunits, which nucleotide sequences are accordingly novel. We have now ascertained the cDNA sequences of the α₂, α₃, α₅, α₆ and β₂ subunits of the human GABA_(A) receptor. These nucleotide sequences, together with the deduced amino acid sequences corresponding thereto, are depicted in FIGS. 2 to 6 of the accompanying drawings. The present invention accordingly provides in several additional aspects DNA molecules encoding the α₂, α₃, α₅, α₆ and β₂ subunits of the human GABA_(A) receptor comprising all or a portion of the sequences depicted in FIGS. 2, 3, 4, 5 and 6 respectively, or substantially similar sequences.

The sequencing of the novel cDNA molecules in accordance with the invention can conveniently be carried out by the standard procedure described in accompanying Example 3; or may be accomplished by alternative molecular cloning techniques which are well known in the art, such as those described by Maniatis et al. in Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, New York, 2nd edition, 1989.

In another aspect, the invention provides a recombinant expression vector comprising the nucleotide sequence of a GABA_(A) receptor subunit together with additional sequences capable of directing the synthesis of the said GABA_(A) receptor subunit in cultures of stably co-transfected eukaryotic cells.

The term "expression vectors" as used herein refers to DNA sequences that are required for the transcription of cloned copies of recombinant DNA sequences or genes and the translation of their mRNAs in an appropriate host. Such vectors can be used to express eukaryotic genes in a variety of hosts such as bacteria, blue-green algae, yeast cells, insect cells, plant cells and animal cells. Specifically designed vectors allow the shuttling of DNA between bacteria-yeast, bacteria-plant or bacteria-animal cells. An appropriately constructed expression vector should contain: an origin of replication for autonomous replication in host cells, selective markers, a limited number of useful restriction enzyme sites, a high copy number, and strong promoters. A promoter is defined as a DNA sequence that directs RNA polymerase to bind to DNA and to initiate RNA synthesis. A strong promoter is one which causes mRNAs to be initiated at high frequency. Expression vectors may include, but are not limited to, cloning vectors, modified cloning vectors, specifically designed plasmids or viruses.

The term "cloning vector" as used herein refers to a DNA molecule, usually a small plasmid or bacteriophage DNA capable of self-replication in a host organism, and used to introduce a fragment of foreign DNA into a host cell. The foreign DNA combined with the vector DNA constitutes a recombinant DNA molecule which is derived from recombinant technology. Cloning vectors may include plasmids, bacteriophages, viruses and cosmids.

The recombinant expression vector in accordance with the invention may be prepared by inserting the nucleotide sequence of the chosen GABA_(A) subunit into a suitable precursor expression vector (hereinafter referred to as the "precursor vector") using conventional recombinant DNA methodology known from the art. The precursor vector may be obtained commercially, or constructed by standard techniques from known expression vectors. The precursor vector suitably contains a selection marker, typically an antibiotic resistance gene, such as the neomycin or ampicillin resistance gene. The precursor vector preferably contains a neomycin resistance gene, adjacent the SV40 early splicing and polyadenylation region; an ampicillin resistance gene; and an origin of replication, e.g. pBR322 ori. The vector also preferably contains an inducible promoter, such as MMTV-LTR (inducible with dexamethasone) or metallothionin (inducible with zinc), so that transcription can be controlled in the cell line of this invention. This reduces or avoids any problem of toxicity in the cells because of the chloride channel intrinsic to the GABA_(A) receptor.

One suitable precursor vector is pMAMneo, available from Clontech Laboratories Inc. (Lee et al., Nature, 1981, 294, 228; and Sardet et al., Cell, 1989, 56, 271). Alternatively the precursor vector pMSGneo can be constructed from the vectors pMSG and pSV2neo as described in Example 1 herein.

The recombinant expression vector of the present invention is then produced by cloning the GABA_(A) receptor subunit cDNA into the above precursor vector. The required receptor subunit cDNA is subcloned from the vector in which it is harboured, and ligated into a restriction enzyme site, e.g. the HindIII site, in the polylinker of the precursor vector, for example pMAMneo or pMSGneo, by standard cloning methodology known from the art, and in particular by techniques analogous to those described in Example 1, step (b) herein. Before this subcloning, it is often advantageous, in order to improve expression, to modify the end of a subunit cDNA with additional 5' untranslated sequences, for example by modifying the 5' end of the γ_(2L) subunit DNA by addition of 5' untranslated region sequences from the α₁ subunit DNA.

One suitable expression vector of the present invention is illustrated in FIG. 1 of the accompanying drawings, in which R represents the nucleotide sequence of a given alpha, beta or gamma subunit of the GABA_(A) receptor, and the remainder of the expression vector depicted therein is derived from the precursor vector pMSGneo and constructed as described in accompanying Example 1, steps (a) and (b).

For each cell line of the present invention, three such vectors will be necessary, one containing an α subunit, one containing a β subunit, and the third containing a γ subunit.

Cells are then co-transfected with the desired combination of three expression vectors. There are several commonly used techniques for transfection of eukaryotic cells in vitro. Calcium phosphate precipitation of DNA is most commonly used (Bachetti et al., Proc. Natl. Acad. Sci. USA, 1977, 74, 1590-1594; Maitland et al., Cell, 1977, 14, 133-141), and represents a favoured technique in the context of the present invention.

A small percentage of the host cells takes up the recombinant DNA. In a small percentage of those, the DNA will integrate into the host cell chromosome. Because the neomycin resistance gene will have been incorporated into these host cells, they can be selected by isolating the individual clones which will grow in the presence of neomycin. Each such clone is then tested to. identify those which will produce the receptor. This is achieved by inducing the production, for example with dexamethasone, and then detecting the presence of receptor by means of radioligand binding.

In a further aspect, the present invention provides protein preparations of GABA_(A) receptor subunit combinations, especially human GABA_(A) receptor subunit combinations, derived from cultures of stably transfected eukaryotic cells. The invention also provides preparations of membranes containing subunit combinations of the GABA_(A) receptor, especially human GABA_(A) receptor subunit combinations, derived from cultures of stably transfected eukaryotic cells. In particular, the protein preparations and membrane preparations according to the invention will suitably contain the α₁ β₁ γ_(2L), α₁ β₃ γ₂, α₂ β₃₁ γ₂, α₅ β₃ γ₂, α₁ β₁ γ_(2S), α₁ β₂ γ₂, α₃ β₃ γ₂ or α₆ β₃ γ₂ subunit combinations of the human GABA_(A) receptor, and will preferably contain a human GABA_(A) receptor consisting of the α₁ β₁ γ_(2L), α₁ β₃ γ_(2S), α₂ β₃ γ_(2S), α₅ β₃ γ_(2S), α₁ β₁ γ_(2S), α₁ β₂ γ_(2S), α₃ β₃ γ_(2S) or α₆ β₃ γ_(2S) subunit combinations. In an especially preferred embodiment, the invention provides cell membranes containing a human GABA_(A) receptor consisting of the α₁ β₁ γ_(2L), α₁ β₃ γ_(2S), α₂ β₃ γ_(2S), α₅ β₃ γ_(2S), α₁ β₁ γ_(2S), α₁ β₂ γ_(2S), α₃ β₃ γ_(2S), or α₆ β₃ γ_(2S) subunit combinations isolated from stably transfected mouse Ltk⁻ fibroblast cells.

The cell line, and the membrane preparations therefrom, according to the present invention have utility in screening and design of drugs which act upon the GABA_(A) receptor, for example benzodiazepines, barbiturates, β-carbolines and neurosteroids. The present invention accordingly provides the use of the cell line described above, and membrane preparations derived therefrom, in screening for and designing medicaments which act upon the GABA_(A) receptor. Of particular interest in this context are molecules capable of interacting selectively with GABA_(A) receptors made up of varying subunit combinations. As will be readily apparent, the cell line in accordance with the present invention, and the membrane preparations derived therefrom, provide ideal systems for the study of structure, pharmacology and function of the various GABA_(A) receptor subtypes.

The following non-limiting Examples illustrate the present invention.

EXAMPLE 1 PREPARATION OF α₁ β₁ γ_(2L) TRANSFECTED CELLS

a) Construction of eukaryotic expression vector pMSGneo

The approx. 2500 base pair HindIII-EcoRI fragment of the vector pMSG (purchased from Pharmacia Biosystems Limited, Milton Keynes, United Kingdom), containing the gpt structural gene and SV40 polyadenylation signals was replaced by the approx. 2800 base pair HindIII-EcoRI fragment of pSV2neo (Southern, P. J. and Berg, P. J., Molecular and Applied Genetics, 1, 327-341, 1982) containing the neomycin resistance gene Neo^(r) and SV40 polyadenylation signals. The EcoRI and HindIII sites were then removed by restriction digesting, blunt ending with klenow polymerase, and religating. EcoRI and HindIII cloning sites were then inserted at the XhoI and SmaI sites of the polylinker by conventional techniques using EcoRI and HindIII linkers.

b) Cloning of subunit cDNAs into pMSGneo

Bovine α₁ and β₁ GABA_(A) receptor cDNAs were obtained from the Molecular Neurobiology Unit, MRC Centre, Hills Road, Cambridge (Scholfield, P. et al. Nature, 328, 221-227, 1987). Bovine γ₂ cDNA was cloned by the method of Whiting, P. et al. (Proc. Natl. Acad. Sci. USA, 87, 9966-9970, 1990). Bovine α₁ was subcloned from pbGRαsense by digestion with EcoRI, blunt ending the DNA with klenow polymerase, addition of HindIII linkers by ligation, digestion with HindIII and ligation into the HindIII site of pMSGneo. Bovine β₁ was subcloned from pbGRβsense by restriction digestion with EcoRI (partial digestion), klenow polymerase blunt ending, ligation of HindIII linkers, restriction digestion with HindIII and ligation into HindIII site of pMSGneo. Before subcloning into pMSGneo, the bovine γ₂ cDNA was modified from the published sequence as follows. The 5' untranslated region of the bovine α₁ cDNA (bases 60-200 of the published sequence) was added to the 5' end of the published γ₂ sequence by amplifying the α₁ untranslated region using polymerase chain reaction, and then subcloning the product into the 5' BamHI (site in the polylinker of the Bluescript Sk⁻ cloning vector; Bluescript vector purchased from Stratagene, San Diego, U.S.A.) HindIII sites of the γ₂ cDNA. The modified γ₂ cDNA was then subcloned into pMSGneo by digestion with XbaI (site in the polylinker of the cloning vector), blunt ending with klenow polymerase, ligation of XhoI linkers, digestion with XhoI (site in the polylinker of the cloning vector), and ligation into XhoI site of pMSGneo.

c) Co-transfection of mouse Ltk⁻ cells

Ltk⁻ cells were obtained from the Salk Institute for Biological Studies, San Diego, Calif. Cells were grown at 37° C., 5-8% CO₂, in Modified Eagles Medium containing penicillin, streptomycin and 10% fetal calf serum. The expression vector harbouring the GABA_(A) receptor subunit DNAs for co-transfection was prepared by a standard protocol (Chen, C. and Okayama, H., BioTechniques, 6, 632-638, 1988). For co-transfection, Ltk⁻ cells were plated in dishes (approx. 2×10⁵ cells/dish) and grown overnight. The transfection was performed by calcium phosphate precipitation using a kit (purchased from 5 Prime→3 Prime Products, Westchester, Pa.). Co-transfection was performed according to manufacturers' instructions, using 5 μg of each subunit DNA construct per 10 cm dish of cells. After 2 days in culture the cells were divided 1:8 into culture medium containing 1 mg/ml neomycin [Geneticin (obtainable from Gibco BRL, Paisley, Scotland, U.K.)]. After a further week the concentration was increased to 1.5 mg/ml, and then 2 mg/ml 1 week after that. Resistant clones of cells were isolated and subcloned using cloning cylinders. Subclones were analysed using radioligand binding: subclones were grown in 10 cm culture dishes, and when confluent changed into culture medium containing 1 μM dexamethasone (obtainable from Sigma Chemical Company, Poole, Dorset, United Kingdom). 3-5 days later the cells were harvested, membranes prepared and used for radioligand binding (see Example 2, step (a) below) using the benzodiazepine antagonist ³ H Ro15-1788 (obtained from New England Nuclear, Du Pont (U.K.) Ltd, Stevenage, United Kingdom). The clone expressing the highest amount of ³ H Ro15-1788 binding was subcloned from a single cell by limiting dilution. The resultant clonal population of cells described below is referred to as population A.

EXAMPLE 2 CHARACTERIZATION OF α₁ β₁ γ_(2L) TRANSFECTED CELLS

a) Radioligand binding

The nature of the recombinant α₁ β₁ γ_(2L) GABA_(A) receptors prepared as described in Example 1 was addressed by characterization of the benzodiazepine (BZ) binding pharmacology, using the BZ antagonist ³ H Ro15-1788. For radioligand binding assays, cells which had been induced by culture in dexamethasone containing medium for 3-5 days were scraped off into 50 mM Tris, pH7.5, 100 mM NaCl in the form of Tris buffered saline (TBS) and pelleted (20,000 rpm, Sorvall RC5C centrifuge). The cell pellet was resuspended in 50 mM Tris, pH7.5, homogenised using an Ultra-Turrax homogeniser and then pelleted as above. This was repeated once more, and the cells then resuspended in TBS (0.4 ml per original 10 cm dish of cells). Radioligand binding was performed in 0.1 ml final volume TBS, containing 5-15 fmols of ³ H Ro15-1788 binding sites. After 1 hour incubation on ice the membranes were harvested onto filters using a Brandel cell harvester, washed with cold TBS, and bound radioactivity determined by scintillation counting. The recombinant α₁ β₁ γ_(2L) receptors bound ³ H Ro15-1788 with high affinity (K_(D) 0.4 nM), at levels of up to 200 fmols/10 cm dish of cells. No binding was seen to either untransfected Ltk⁻ cells, or population A cells which had not been induced by addition of dexamethasone to the culture medium, confirming that the ³ H Ro15-1788 was binding to recombinant α₁ β₁ γ₂ GABA_(A) receptors. The ³ H Ro15-1788 binding was inhibited by flunitrazepam, CL218872, FG8205, βCCM, zolpidem and Ro15-4513, confirming the BZ pharmacology of the recombinant receptor. Since it is established that only GABA_(A) receptors containing an α, a β and a γ subunit exhibit BZ binding (Pritchett, D. et al., Nature, 338, 582-585, 1989) these data confirm the nature of the recombinant α₁ β₁ γ₂ GABA_(A) receptors expressed by population A cells.

b) Electrophysiology

The nature of the GABA_(A) receptor expressed by population A cells has been extensively characterised by electrophysiological techniques, using whole cell patch clamp. Only cells induced by culture in the presence of dexamethasone showed responses to GABA. Concentration response curves to GABA gave a log EC₅₀ of 5.2, and a Hill coefficient of 1.9. The response to GABA was potentiated by BZs flunitrazepam and CL218872, by the barbiturate pentobarbitone, and by the steroid alphaxalone. The response to GABA was antagonised by both bicuculline and picrotoxin. All these electrophysiological data confirm that the recombinant GABA_(A) receptor expressed by population A cells has all of the properties expected of a bona fide GABA_(A) receptor.

EXAMPLE 3 ISOLATION AND SEQUENCING OF cDNAS ENCODING HUMAN GABA_(A) RECEPTOR α₂, α₃, α₅, α₆ & β₂ SUBUNITS

a) cDNA libraries

cDNAs were cloned from human foetal brain (α₂, α₃), hippocampal (α₅, β₂) and cerebellum (α₆) lambda bacteriophage cDNA libraries. All cDNA libraries were constructed in the lambdaZAP vector, and were purchased from Stratagene (San Diego, Calif.). For screening, the cDNA libraries were plated according to the manufacturer's instructions, at 40,000 pfu per 137 mm plate. Filter lifts were taken using Hybond N filters (Amersham) according to the manufacturer's instructions.

b) Isolation of cDNA encoding human α₂ subunit

A bovine α₂ cDNA (obtained from E. Barnard, Molecular Neurobiology, University of Cambridge, Hills Road, Cambridge; Levitan et al., Nature, 1988, 335, 76) was labelled to high specific activity (>1.10⁹ cpm/μg) with ³² P by random priming and used as a probe. Library filters (8 replica filters) were prehybridised for 3-6 hours at 42° C. in 5× SSPE (1× SSPE is 0.18M NaCl, 0.01M Na₃ PO₄ [pH7.4], 1 mM EDTA), 5× Denhardt's solution, 100 μg/ml salmon sperm DNA, 0.1% sodium dodecyl sulphate (SDS), 30% formamide. Hybridisation was performed in the same buffer for 18 hours at 42° C., including 0.5-1.10⁶ cpm ³² P-labelled probe per ml of hybridisation buffer. Filters were washed at 55° C. in 5× SSPE (2× 15 minutes) and 1× SSPE (2× 15 minutes) and exposed to Kodak XAR film for 1-3 days. Positive clones were plaque purified using standard techniques, and the Bluescript plasmid (Stratagene) "rescued" according to manufacturer's instructions. cDNA clones were sequenced on both strands by standard techniques using Sequenase II enzyme (United States Biochemicals). The nucleotide sequence of the cDNA encoding the human GABA_(A) receptor α₂ subunit, together with the deduced amino acid sequence corresponding thereto, is shown in FIG. 2 of the accompanying drawings.

c) Isolation of cDNA encoding human α₃ subunit

A bovine α₃ cDNA (obtained from E. Barnard, Molecular Neurobiology, University of Cambridge, Hills Road, Cambridge; Levitan et al., Nature, 1988, 335, 76) was labelled to high specific activity with ³² P by random priming and used as a probe. Library filters were prehybridised for 3-6 hours at 55° C. in 5× SSPE, 5× Denhardt's solution, 0.1% SDS, 100 μg/ml salmon sperm DNA, and hybridised for 18 hours, 55° C. in the same buffer, containing 0.5-1×10⁶ cpm/ml of ³² P-labelled bovine α₃ cDNA as probe. Filters were washed and exposed to X-ray film as described above; cDNA clones were rescued and sequenced as described above. The longest α₃ cDNA clone was missing in approximately 100 bp of the 5' end of the coding region. This was obtained by PCR using as primers an oligonucleotide "anchor" primer derived from the T7 primer sequence of Bluescript vector (5'AGCGCGCGTAATACGACTCACTATAGGGCGAA3'; SEQ ID NO:11) and an oligonucleotide derived from sequence near the 5' end of the truncated α₃ cDNA, containing an internal Hpal site (5'CAGCATGAATTGTTAACCTCATTGTA3'; SEQ ID NO:12). Oligonucleotides were synthesised on an Applied Biosystems 380B synthesiser. PCR was performed as described above, and a 300 bp PCR product obtained which was double digested with Hpal and Kpnl and subcloned into the similarly cut truncated α₃ cDNA to yield a full length human α₃ cDNA. The cDNA was sequenced on both strands as described above. The nucleotide sequence of the cDNA encoding the human GABA_(A) receptor α₃ subunit, together with the deduced amino acid sequence corresponding thereto, is shown in FIG. 3 of the accompanying drawings.

d) Isolation of cDNA encoding human α₅ subunit

A rat α₅ cDNA obtained by polymerase chain reaction (PCR) was used as a probe to screen the cDNA library. For PCR, sequences of the oligonucleotide primers were taken from the published α₅ sequences (Khrestchatisky et al., Neuron, 1989, 3, 745) and incorporated a Hind III site for subcloning purposes: 5'ATTATTCAAGCTTGCCATGGACAATGGAATGCTC3'[SEQ ID NO:13] (bp114-148); 5'GGTTTCCAGCTTACTTTGGAGAGGTAGC3'[SEQ ID NO;14] (bp1507-1535). PCR and subcloning of the PCR product into Bluescript SK-vector (Stratagene) for analysis was performed as described elsewhere (Whiting et al., Proc. Natl. Acad. Sci. USA, 1990, 87, 9966) except that rat brain cDNA was used as template. The rat α₅ cDNA was labelled with ³² P and used to screen the human hippocampal cDNA library, and positive α₅ clones rescued and sequenced as described for α₂ above. The nucleotide sequence of the cDNA encoding the human GABA_(A) receptor α₅ subunit, together with the deduced amino acid sequence corresponding thereto, is shown in FIG. 4 of the accompanying drawings.

e) Isolation of cDNA encoding human α₆ subunit

A rat α₆ cDNA obtained by PCR was used as a probe to screen the cDNA library. PCR was performed as described above for α₅, using oligonucleotide primers derived from the published rat α₆ sequence (Luddens et al., Nature, 1990, 346, 648) incorporating an EcoRI site for subcloning purposes: 5'GAGGAAGAATTCAGGAGGGTGACCT3'[SEQ ID NO:15] (bp48-72); 5'GAAAATAACGAATTCCAGTGTCCAGCTTT3'[SEQ ID NO:16] (bp1376-1404). The rat α₆ cDNA clone isolated by PCR was labelled with ³² P and used to screen a human cerebellum cDNA library, as described above for α₂. Positive α₆ clones were purified, rescued and sequenced as described above. None of the cDNAs contained a complete coding region. To obtain a full length cDNA 3 clones were joined together using convenient restriction sites. The nucleotide sequence of the cDNA encoding the human GABA_(A) receptor α₆ subunit, together with the deduced amino acid sequence corresponding thereto, is shown in FIG. 5 of the accompanying drawings.

f) Isolation of cDNA encoding human α₂ subunit

Human β₂ cDNA was isolated using as a probe a short human β₂ cDNA obtained by PCR. PCR was performed as described above (except that the human cerebellum cDNA library was used as template), using oligonucleotide primers derived from the published rat β₂ sequence (Ymer et al., EMBO J., 1989, 8, 1665), incorporating EcoRI sites for subcloning purposes: 5'CAAAAGAATTCAGCTGAGAAAGCTGCTAATGC3'[SEQ ID NO:17] (bp1088-1119); 5'TCAGGCGAATTCTCTTTTGTGCCACATGTCGTTC3'[SEQ ID NO:18] (bp1331-1364). The human β₂ clone obtained by PCR was radiolabelled with ³² P and used to screen a human hippocampal cDNA library, as described above for α₂. The largest cDNA clone obtained lacked the 5' 500 bp of the coding region of the β₂ subunit. This was obtained by PCR using as primers an oligonucleotide "anchor" primer derived from the T7 primer sequence of the Bluescript vector (5'AGCGCGCGTAATACGACTCACTATAGGGCGAA3'; SEQ ID NO:19), and an oligonucleotide derived from sequence near the 5' end of the truncated β₂ cDNA, containing a Kpnl site (5'CATCCAGTGGGTACCTCCTTAGGT3'; SEQ ID NO:20). PCR was performed as described above, and a 700 bp PCR product obtained which was digested with kpnl and subcloned into the truncated cDNA clone (also Kpnl digested) to yield a full length human β₂ cDNA. The nucleotide sequence of the cDNA encoding the human GABA_(A) receptor β₂ subunit, together with the deduced amino acid sequence corresponding thereto, is shown in FIG. 6 of the accompanying drawings.

EXAMPLE 4 PREPARATION OF STABLY TRANSFECTED CELLS EXPRESSING α₁ β₃ γ_(2S), α₂ β₃ γ_(2S) AND α₅ β₃ γ_(2S) SUBUNIT COMBINATIONS OF THE HUMAN GABA_(A) RECEPTOR

Isolation and sequence of human α₂ and α₅ cDNAs have been described in Example 3. The sequence of human α₁ cDNA has been published previously by Schofield et al., FEBS Lett., 1989, 244, 361. It differs from the bovine sequence at a single amino acid (trp95 in bovine α₁ ; arg in human α₁). To create a human α₁ cDNA the bovine sequence was converted to the human by site directed mutagenesis of amino acid 95 with the oligonucleotide 5'GCAATGAAAATCCGGACTGGCAT3'; SEQ ID NO:21), using methods described elsewhere (K. Wafford and P. Whiting, FEBS Lett., 1992, 313, 113-117). The sequence of human γ₂ has been published previously by Pritchett et al., Nature, 1989, 338, 582. A human γ₂ cDNA was isolated by PCR using conditions described elsewhere (Whiting et al., Proc. Natl. Acad. Sci. USA, 1990, 87, 9966-9970), using human hippocampal cDNA library as template and oligonucleotide primers derived from the 5' and 3' untranslated regions of the published γ₂ sequence, incorporating a Hind III restriction site:

5'GGGAGGGAAGCTTCTGCAACCAAGAGGC3'; SEQ ID NO:22),

5'ACCACATAGAAGCTTATTTAAGTGGAC3'; SEQ ID NO:23). Sequencing indicated that the form of γ₂ used is the short form, γ_(2S), lacking the 24 bp insert in the putative cytoplasmic loop region (Whiting et al., Proc. Natl. Acad. Sci. USA, 1990, 87, 9966-9970). The sequence of human β₃ has been published by Wagstaff et al., Am. J. Hum. Genet., 1991, 41, 330-337. A human β₃ cDNA was isolated by screening a human foetal brain cDNA library (see Example 3) with a short human β₃ cDNA probe encoding the putative cytoplasmic loop domain which had been obtained using PCR.

Human α₁, α₂, α₅, β₃ and γ_(2S) cDNAs were subcloned into the eukaryotic expression vector pMSGneo (see Example 1) using standard techniques (cf. Maniatis et al. in Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, New York, 2nd Edition, 1989) and stable cell lines expressing human α₁ β₃ γ_(2S), α₂ β₃ γ_(2S) and α₅ β₃ γ_(2S) GABA_(A) receptors were established as described in Example 1.

EXAMPLE 5 PREPARATION OF STABLY TRANSFECTED CELLS EXPRESSING α₁ β₁ γ_(2S), α₁ β₂ γ_(2S), α₃ β₃ γ_(2S) AND α₆ β₃ γ_(2S) SUBUNIT COMBINATIONS OF THE HUMAN GABA_(A) RECEPTOR

Isolation of α₃ and α₆ cDNAs is as described in Example 3, and isolation of α₁, β₃ and γ_(2S) cDNAs is as described above in Example 4. Human β₁ subunit cDNA was isolated by PCR from human brain cDNA as described above. Oligonucleotide primers used for the PCR were derived from the published human β₁ sequence (Schofield et al., FEBS Lett., 1989, 244, 361-364), 5' and 3' untranslated regions incorporating Hind III restriction enzyme sites for subcloning:

5'TAATCAAGCTTAGTAATGTGGACAGTACAAAAT3'; SEQ ID NO:24) and

5'AAATGGAAGCTTTAGAACAGACCTCAGTGTACA3'; SEQ ID NO:25). Human α₁, α₃, α₆, β₁, β₂, β₃ and γ_(2S) cDNAs were subcloned into the eukaryotic expression vector pMSGneo (see Example 1) using standard techniques (cf. Maniatis et al. in Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, New York, 2nd Edition, 1989) and stable cell lines expressing human α₁ β₁ γ_(2S), α₁ β₂ γ_(2S), α₃ β₃ γ_(2S) and α₆ β₃ γ_(2S) GABA_(A) receptors were established as described in Example 1.

    __________________________________________________________________________     SEQUENCE LISTING     (1) GENERAL INFORMATION:     (iii) NUMBER OF SEQUENCES: 25     (2) INFORMATION FOR SEQ ID NO: 1:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 2310 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: cDNA     (ix) FEATURE:     (A) NAME/KEY: CDS     (B) LOCATION: 298..1683     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:     GAATTCCCCCCTTGCAGGCCGAGCCGGGGCCCTGCGCCCTCCCCCTCCGCCCAGCTCGGC60     CAAGGGCGCATTTGCTGAGCGTCTGGCGGCCTCTACCGGAGCACCTCTGCAGAGGGCCGA120     TCCTCCAGCCCAGAGACGACATGTGGCGCTCGGGCGAGTGCCTTGCAGAGAGAGGAGTAG180     CTTGCTGGCTTTGAACGCGTGGCGTGGCAGATATTTCAGAAAGCTTCAAGAACAAGCTGG240     AGAAGGGAAGAGTTATTCCTCCATATTCACCTGCTTCAACTACTATTCTTATTGGGA297     ATGGACAATGGAATGTTCTCTGGTTTTATCATGATCAAAAACCTCCTT345     MetAspAsnGlyMetPheSerGlyPheIleMetIleLysAsnLeuLeu     151015     CTCTTTTGTATTTCCATGAACTTATCCAGTCACTTTGGCTTTTCACAG393     LeuPheCysIleSerMetAsnLeuSerSerHisPheGlyPheSerGln     202530     ATGCCAACCAGTTCAGTGAAAGATGAGACCAATGACAACATCACGATA441     MetProThrSerSerValLysAspGluThrAsnAspAsnIleThrIle     354045     TTTACCAGGATCTTGGATGGGCTCTTGGATGGCTACGACAACAGACTT489     PheThrArgIleLeuAspGlyLeuLeuAspGlyTyrAspAsnArgLeu     505560     CGGCCCGGGCTGGGAGAGCGCATCACTCAGGTGAGGACCGACATCTAC537     ArgProGlyLeuGlyGluArgIleThrGlnValArgThrAspIleTyr     65707580     GTCACCAGCTTCGGCCCGGTGTCCGACACGGAAATGGAGTACACCATA585     ValThrSerPheGlyProValSerAspThrGluMetGluTyrThrIle     859095     GACGTGTTTTTCCGACAAAGCTGGAAAGATGAAAGGCTTCGGTTTAAG633     AspValPhePheArgGlnSerTrpLysAspGluArgLeuArgPheLys     100105110     GGGCCCATGCAGCGCCTCCCTCTCAACAACCTCCTTGCCAGCAAGATC681     GlyProMetGlnArgLeuProLeuAsnAsnLeuLeuAlaSerLysIle     115120125     TGGACCCCAGACACGTTCTTCCACAACGGGAAGAAGTCCATCGCTCAC729     TrpThrProAspThrPhePheHisAsnGlyLysLysSerIleAlaHis     130135140     AACATGACCACGCCCAACAAGCTGCTGCGGCTGGAGGACGACGGCACC777     AsnMetThrThrProAsnLysLeuLeuArgLeuGluAspAspGlyThr     145150155160     CTGCTCTACACCATGCGCTTGACCATCTCTGCAGAGTGCCCCATGCAG825     LeuLeuTyrThrMetArgLeuThrIleSerAlaGluCysProMetGln     165170175     CTTGAGGACTTCCCGATGGATGCGCACGCTTGCCCTCTGAAATTTGGC873     LeuGluAspPheProMetAspAlaHisAlaCysProLeuLysPheGly     180185190     AGCTATGCGTACCCTAATTCTGAAGTCGTTTACGTCTGGACCAACGGC921     SerTyrAlaTyrProAsnSerGluValValTyrValTrpThrAsnGly     195200205     TCCACCAAGTCGGTGGTGGTGGCGGAAGATGGCTCCAGACTGAACCAG969     SerThrLysSerValValValAlaGluAspGlySerArgLeuAsnGln     210215220     TACCACCTGATGGGGCAGACGGTGGGCACTGAGAACATCAGCACCAGC1017     TyrHisLeuMetGlyGlnThrValGlyThrGluAsnIleSerThrSer     225230235240     ACAGGCGAATACACAATCATGACAGCTCACTTCCACCTGAAAAGGAAG1065     ThrGlyGluTyrThrIleMetThrAlaHisPheHisLeuLysArgLys     245250255     ATTGGCTACTTTGTCATCCAGACCTACCTTCCCTGCATAATGACCGTG1113     IleGlyTyrPheValIleGlnThrTyrLeuProCysIleMetThrVal     260265270     ATCTTATCACAGGTGTCCTTTTGGCTGAACCGGGAATCAGTCCCAGCC1161     IleLeuSerGlnValSerPheTrpLeuAsnArgGluSerValProAla     275280285     AGGACAGTTTTTGGGGTCACCACGGTGCTGACCATGACGACCCTCAGC1209     ArgThrValPheGlyValThrThrValLeuThrMetThrThrLeuSer     290295300     ATCAGCGCCAGGAACTCTCTGCCCAAAGTGGCCTACGCCACCGCCATG1257     IleSerAlaArgAsnSerLeuProLysValAlaTyrAlaThrAlaMet     305310315320     GACTGGTTCATAGCTGTGTGCTATGCCTTCGTCTTCTCGGCGCTGATA1305     AspTrpPheIleAlaValCysTyrAlaPheValPheSerAlaLeuIle     325330335     GAGTTTGCCACGGTCAATTACTTTACCAAGAGAGGCTGGGCCTGGGAT1353     GluPheAlaThrValAsnTyrPheThrLysArgGlyTrpAlaTrpAsp     340345350     GGCAAAAAAGCCTTGGAAGCAGCCAAGATCAAGAAAAAGCGTGAAGTC1401     GlyLysLysAlaLeuGluAlaAlaLysIleLysLysLysArgGluVal     355360365     ATACTAAATAAGTCAACAAACGCTTTTACAACTGGGAAGATGTCTCAC1449     IleLeuAsnLysSerThrAsnAlaPheThrThrGlyLysMetSerHis     370375380     CCCCCAAACATTCCGAAGGAACAGACCCCAGCAGGGACGTCGAATACA1497     ProProAsnIleProLysGluGlnThrProAlaGlyThrSerAsnThr     385390395400     ACCTCAGTCTCAGTAAAACCCTCTGAAGAGAAGACTTCTGAAAGCAAA1545     ThrSerValSerValLysProSerGluGluLysThrSerGluSerLys     405410415     AAGACTTACAACAGTATCAGCAAAATTGACAAAATGTCCCGAATCGTA1593     LysThrTyrAsnSerIleSerLysIleAspLysMetSerArgIleVal     420425430     TTCCCAGTCTTGTTCGGCACTTTCAACTTAGTTTACTGGGCAACGTAT1641     PheProValLeuPheGlyThrPheAsnLeuValTyrTrpAlaThrTyr     435440445     TTGAATAGGGAGCCGGTGATAAAAGGAGCCGCCTCTCCAAAA1683     LeuAsnArgGluProValIleLysGlyAlaAlaSerProLys     450455460     TAACCGGCCACACTCCCAAACTCCAAGACAGCCATACTTCCAGCGAAATGGTACCAAGGA1743     GAGGTTTTGCTCACAGGGACTCTCCATATGTGAGCACTATCTTTCAGGAAATTTTTGCAT1803     GTTTAATAATATGTACAAATAATATTGCCTTGATGTTTCTATATGTAACTTCAGATGTTT1863     CCAAGATGTCCCATTGATAATTCGAGCAAACAACTTTCTGGAAAAACAGGATACGATGAC1923     TGACACTCAGATGCCCAGTATCATACGTTGATAGTTTACAAACAAGATACGTATATTTTT1983     AACTGCTTCAAGTGTTACCTAACAATGTTTTTTATACTTCAAATGTCATTTCATACAAAT2043     TTTCCCAGTGAATAAATATTTTAGGAAACTCTCCATGATTATTAGAAGACCAACTATATT2103     GCGAGAAACAGAGATCATAAAGAGCACGTTTTCCATTATGAGGAAACTTGGACATTTATG2163     TACAAAATGAATTGCCTTTGATAATTCTTACTGTTCTGAAATTAGGAAAGTACTTGCATG2223     ATCTTACACGAAGAAATAGAATAGGCAAACTTTTATGTAGGCAGATTAATAACAGAAATA2283     CATCATATGTTAGATACACAAAATATT2310     (2) INFORMATION FOR SEQ ID NO: 2:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 462 amino acids     (B) TYPE: amino acid     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: protein     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:     MetAspAsnGlyMetPheSerGlyPheIleMetIleLysAsnLeuLeu     151015     LeuPheCysIleSerMetAsnLeuSerSerHisPheGlyPheSerGln     202530     MetProThrSerSerValLysAspGluThrAsnAspAsnIleThrIle     354045     PheThrArgIleLeuAspGlyLeuLeuAspGlyTyrAspAsnArgLeu     505560     ArgProGlyLeuGlyGluArgIleThrGlnValArgThrAspIleTyr     65707580     ValThrSerPheGlyProValSerAspThrGluMetGluTyrThrIle     859095     AspValPhePheArgGlnSerTrpLysAspGluArgLeuArgPheLys     100105110     GlyProMetGlnArgLeuProLeuAsnAsnLeuLeuAlaSerLysIle     115120125     TrpThrProAspThrPhePheHisAsnGlyLysLysSerIleAlaHis     130135140     AsnMetThrThrProAsnLysLeuLeuArgLeuGluAspAspGlyThr     145150155160     LeuLeuTyrThrMetArgLeuThrIleSerAlaGluCysProMetGln     165170175     LeuGluAspPheProMetAspAlaHisAlaCysProLeuLysPheGly     180185190     SerTyrAlaTyrProAsnSerGluValValTyrValTrpThrAsnGly     195200205     SerThrLysSerValValValAlaGluAspGlySerArgLeuAsnGln     210215220     TyrHisLeuMetGlyGlnThrValGlyThrGluAsnIleSerThrSer     225230235240     ThrGlyGluTyrThrIleMetThrAlaHisPheHisLeuLysArgLys     245250255     IleGlyTyrPheValIleGlnThrTyrLeuProCysIleMetThrVal     260265270     IleLeuSerGlnValSerPheTrpLeuAsnArgGluSerValProAla     275280285     ArgThrValPheGlyValThrThrValLeuThrMetThrThrLeuSer     290295300     IleSerAlaArgAsnSerLeuProLysValAlaTyrAlaThrAlaMet     305310315320     AspTrpPheIleAlaValCysTyrAlaPheValPheSerAlaLeuIle     325330335     GluPheAlaThrValAsnTyrPheThrLysArgGlyTrpAlaTrpAsp     340345350     GlyLysLysAlaLeuGluAlaAlaLysIleLysLysLysArgGluVal     355360365     IleLeuAsnLysSerThrAsnAlaPheThrThrGlyLysMetSerHis     370375380     ProProAsnIleProLysGluGlnThrProAlaGlyThrSerAsnThr     385390395400     ThrSerValSerValLysProSerGluGluLysThrSerGluSerLys     405410415     LysThrTyrAsnSerIleSerLysIleAspLysMetSerArgIleVal     420425430     PheProValLeuPheGlyThrPheAsnLeuValTyrTrpAlaThrTyr     435440445     LeuAsnArgGluProValIleLysGlyAlaAlaSerProLys     450455460     (2) INFORMATION FOR SEQ ID NO: 3:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 1408 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: cDNA     (ix) FEATURE:     (A) NAME/KEY: CDS     (B) LOCATION: 27..1385     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:     AATTCTGCATTTCAGTGCACTGCAGGATGGCGTCATCTCTGCCCTGGCTGTGC53     MetAlaSerSerLeuProTrpLeuCys     15     ATTATTCTGTGGCTAGAAAATGCCCTAGGGAAACTCGAAGTTGAAGGC101     IleIleLeuTrpLeuGluAsnAlaLeuGlyLysLeuGluValGluGly     10152025     AACTTCTACTCAGAAAACGTCAGTCGGATCCTGGACAACTTGCTTGAA149     AsnPheTyrSerGluAsnValSerArgIleLeuAspAsnLeuLeuGlu     303540     GGCTATGACAATCGGCTGCGGCCGGGATTTGGAGGTGCTGTCACTGAA197     GlyTyrAspAsnArgLeuArgProGlyPheGlyGlyAlaValThrGlu     455055     GTCAAAACAGACATTTATGTGACCAGTTTTGGGCCCGTGTCAGATGTG245     ValLysThrAspIleTyrValThrSerPheGlyProValSerAspVal     606570     GAGATGGAGTATACGATGGATGTTTTTTTTCGCCAGACCTGGACTGAT293     GluMetGluTyrThrMetAspValPhePheArgGlnThrTrpThrAsp     758085     GAGAGGTTGAAGTTTGGGGGGCCAACTGAGATTCTGAGTCTGAATAAT341     GluArgLeuLysPheGlyGlyProThrGluIleLeuSerLeuAsnAsn     9095100105     TTGATGGTCAGTAAAATCTGGACGCCTGACACCTTTTTCAGAAATGGT389     LeuMetValSerLysIleTrpThrProAspThrPhePheArgAsnGly     110115120     AAAAAGTCCATTGCTCACAACATGACAACTCCTAATAAACTCTTCAGA437     LysLysSerIleAlaHisAsnMetThrThrProAsnLysLeuPheArg     125130135     ATAATGCAGAATGGAACCATTTTATACACCATGAGGCTTACCATCAAT485     IleMetGlnAsnGlyThrIleLeuTyrThrMetArgLeuThrIleAsn     140145150     GCTGACTGTCCCATGAGGCTGGTTAACTTTCCTATGGATGGGCATGCT533     AlaAspCysProMetArgLeuValAsnPheProMetAspGlyHisAla     155160165     TGTCCACTCAAGTTTGGGAGCTATGCTTATCCCAAAAGTGAAATCATA581     CysProLeuLysPheGlySerTyrAlaTyrProLysSerGluIleIle     170175180185     TATACGTGGAAAAAAGGACCACTTTACTCAGTAGAAGTCCCAGAAGAA629     TyrThrTrpLysLysGlyProLeuTyrSerValGluValProGluGlu     190195200     TCTTCAAGCCTTCTCCAGTATGATCTGATTGGACAAACAGTATCTAGT677     SerSerSerLeuLeuGlnTyrAspLeuIleGlyGlnThrValSerSer     205210215     GAGACAATTAAATCTAACACAGGTGAATACGTTATAATGACAGTTTAC725     GluThrIleLysSerAsnThrGlyGluTyrValIleMetThrValTyr     220225230     TTCCACTTGCAAAGGAAGATGGGCTACTTCATGATACAGATATACACT773     PheHisLeuGlnArgLysMetGlyTyrPheMetIleGlnIleTyrThr     235240245     CCTTGCATTATGACAGTCATTCTTTCCCAGGTGTCTTTCTGGATTAAT821     ProCysIleMetThrValIleLeuSerGlnValSerPheTrpIleAsn     250255260265     AAGGAGTCCGTCCCAGCAAGAACTGTTCTTGGGATCACCACTGTTTTA869     LysGluSerValProAlaArgThrValLeuGlyIleThrThrValLeu     270275280     ACTATGACCACTTTGAGCATCAGTGCCCGGCACTCTTTGCCAAAAGTG917     ThrMetThrThrLeuSerIleSerAlaArgHisSerLeuProLysVal     285290295     TCATATGCCACTGCCATGGATTGGTTCATAGCTGTTTGCTTTGCATTC965     SerTyrAlaThrAlaMetAspTrpPheIleAlaValCysPheAlaPhe     300305310     GTCTTCTCTGCTCTTATCGAGTTCGCAGCTGTCAACTACTTTACCAAT1013     ValPheSerAlaLeuIleGluPheAlaAlaValAsnTyrPheThrAsn     315320325     CTTCAGACACAGAAGGCGAAAAGGAAGGCACAGTTTGCAGCCCCACCC1061     LeuGlnThrGlnLysAlaLysArgLysAlaGlnPheAlaAlaProPro     330335340345     ACAGTGACAATATCAAAAGCTACTGAACCTTTGGAAGCTGAGATTGTT1109     ThrValThrIleSerLysAlaThrGluProLeuGluAlaGluIleVal     350355360     TTGCATCCTGACTCCAAATATCATCTGAAGAAAAGGATCACTTCTCTG1157     LeuHisProAspSerLysTyrHisLeuLysLysArgIleThrSerLeu     365370375     TCTTTGCCAATAGTTTCATCTTCCGAGGCCAATAAAGTGCTCACGAGA1205     SerLeuProIleValSerSerSerGluAlaAsnLysValLeuThrArg     380385390     GCGCCCATCTTACAATCAACACCTGTCACACCCCCACCACTCCCGCCA1253     AlaProIleLeuGlnSerThrProValThrProProProLeuProPro     395400405     GCCTTTGGAGGCACCAGTAAAATAGACCAGTATTCTCGAATTCTCTTC1301     AlaPheGlyGlyThrSerLysIleAspGlnTyrSerArgIleLeuPhe     410415420425     CCAGTTGCATTTGCAGGATTCAACCTTGTGTACTGGGTAGTTTATCTT1349     ProValAlaPheAlaGlyPheAsnLeuValTyrTrpValValTyrLeu     430435440     TCCAAAGATACAATGGAAGTGAGTAGCAGTGTTGAATAGCTTTTCC1395     SerLysAspThrMetGluValSerSerSerValGlu     445450     AGGACAACCTGAA1408     (2) INFORMATION FOR SEQ ID NO: 4:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 453 amino acids     (B) TYPE: amino acid     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: protein     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:     MetAlaSerSerLeuProTrpLeuCysIleIleLeuTrpLeuGluAsn     151015     AlaLeuGlyLysLeuGluValGluGlyAsnPheTyrSerGluAsnVal     202530     SerArgIleLeuAspAsnLeuLeuGluGlyTyrAspAsnArgLeuArg     354045     ProGlyPheGlyGlyAlaValThrGluValLysThrAspIleTyrVal     505560     ThrSerPheGlyProValSerAspValGluMetGluTyrThrMetAsp     65707580     ValPhePheArgGlnThrTrpThrAspGluArgLeuLysPheGlyGly     859095     ProThrGluIleLeuSerLeuAsnAsnLeuMetValSerLysIleTrp     100105110     ThrProAspThrPhePheArgAsnGlyLysLysSerIleAlaHisAsn     115120125     MetThrThrProAsnLysLeuPheArgIleMetGlnAsnGlyThrIle     130135140     LeuTyrThrMetArgLeuThrIleAsnAlaAspCysProMetArgLeu     145150155160     ValAsnPheProMetAspGlyHisAlaCysProLeuLysPheGlySer     165170175     TyrAlaTyrProLysSerGluIleIleTyrThrTrpLysLysGlyPro     180185190     LeuTyrSerValGluValProGluGluSerSerSerLeuLeuGlnTyr     195200205     AspLeuIleGlyGlnThrValSerSerGluThrIleLysSerAsnThr     210215220     GlyGluTyrValIleMetThrValTyrPheHisLeuGlnArgLysMet     225230235240     GlyTyrPheMetIleGlnIleTyrThrProCysIleMetThrValIle     245250255     LeuSerGlnValSerPheTrpIleAsnLysGluSerValProAlaArg     260265270     ThrValLeuGlyIleThrThrValLeuThrMetThrThrLeuSerIle     275280285     SerAlaArgHisSerLeuProLysValSerTyrAlaThrAlaMetAsp     290295300     TrpPheIleAlaValCysPheAlaPheValPheSerAlaLeuIleGlu     305310315320     PheAlaAlaValAsnTyrPheThrAsnLeuGlnThrGlnLysAlaLys     325330335     ArgLysAlaGlnPheAlaAlaProProThrValThrIleSerLysAla     340345350     ThrGluProLeuGluAlaGluIleValLeuHisProAspSerLysTyr     355360365     HisLeuLysLysArgIleThrSerLeuSerLeuProIleValSerSer     370375380     SerGluAlaAsnLysValLeuThrArgAlaProIleLeuGlnSerThr     385390395400     ProValThrProProProLeuProProAlaPheGlyGlyThrSerLys     405410415     IleAspGlnTyrSerArgIleLeuPheProValAlaPheAlaGlyPhe     420425430     AsnLeuValTyrTrpValValTyrLeuSerLysAspThrMetGluVal     435440445     SerSerSerValGlu     450     (2) INFORMATION FOR SEQ ID NO: 5:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 1866 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: cDNA     (ix) FEATURE:     (A) NAME/KEY: CDS     (B) LOCATION: 225..1646     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:     GAATTCCGCGCGGGGAAGGGAAGAAGAGGACGAGGTGGCGCAGAGACCGCGGGAGAACAC60     AGTGCCTCCGGAGGAAATCTGCTCGGTCCCCGGCAGCCGCGCTTCCCCTTTGATGTTTTG120     GTACGCCGTGGCCATGCGCCTCACATTAGAATTACTGCACTGGGCAGACTAAGTTGGATC180     TCCTCTCTTCAGTGAAACCCTCAATTCCATCAAAAACTAAAGGGATGTGGAGAGTG236     MetTrpArgVal     CGGAAAAGGGGCTACTTTGGGATTTGGTCCTTCCCCTTAATAATCGCC284     ArgLysArgGlyTyrPheGlyIleTrpSerPheProLeuIleIleAla     5101520     GCTGTCTGTGCGCAGAGTGTCAATGACCCTAGTAATATGTCGCTGGTT332     AlaValCysAlaGlnSerValAsnAspProSerAsnMetSerLeuVal     253035     AAAGAGACGGTGGATAGACTCCTGAAAGGCTATGACATTCGTCTGAGA380     LysGluThrValAspArgLeuLeuLysGlyTyrAspIleArgLeuArg     404550     CCAGATTTTGGAGGTCCCCCCGTGGCTGTGGGGATGAACATTGACATT428     ProAspPheGlyGlyProProValAlaValGlyMetAsnIleAspIle     556065     GCCAGCATCGATATGGTTTCTGAAGTCAATATGGATTATACCTTGACA476     AlaSerIleAspMetValSerGluValAsnMetAspTyrThrLeuThr     707580     ATGTACTTTCAACAAGCCTGGAGAGATAAGAGGCTGTCCTATAATGTA524     MetTyrPheGlnGlnAlaTrpArgAspLysArgLeuSerTyrAsnVal     859095100     ATACCTTTAAACTTGACTCTGGACAACAGAGTGGCAGACCAGCTCTGG572     IleProLeuAsnLeuThrLeuAspAsnArgValAlaAspGlnLeuTrp     105110115     GTGCCTGATACCTATTTCCTGAACGATAAGAAGTCATTTGTGCACGGA620     ValProAspThrTyrPheLeuAsnAspLysLysSerPheValHisGly     120125130     GTGACTGTTAAGAACCGCATGATTCGCCTGCATCCTGATGGCACCGTC668     ValThrValLysAsnArgMetIleArgLeuHisProAspGlyThrVal     135140145     CTTTATGGACTCAGAATCACAACCACAGCTGCCTGCATGATGGACCTA716     LeuTyrGlyLeuArgIleThrThrThrAlaAlaCysMetMetAspLeu     150155160     AGGAGGTACCCACTGGATGAACAAAACTGCACCTTGGAAATTGAGAGC764     ArgArgTyrProLeuAspGluGlnAsnCysThrLeuGluIleGluSer     165170175180     TATGGATACACAACTGATGACATTGAGTTTTACTGGCGTGGCGATGAT812     TyrGlyTyrThrThrAspAspIleGluPheTyrTrpArgGlyAspAsp     185190195     AATGCAGTAACAGGAGTAACGAAAATTGAACTTCCACAGTTCTCTATT860     AsnAlaValThrGlyValThrLysIleGluLeuProGlnPheSerIle     200205210     GTAGATTACAAACTTATCACCAAGAAGGTTGTTTTTTCCACAGGTTCC908     ValAspTyrLysLeuIleThrLysLysValValPheSerThrGlySer     215220225     TATCCCAGGTTATCCCTCAGCTTTAAGCTTAAGAGAAACATTGGCTAC956     TyrProArgLeuSerLeuSerPheLysLeuLysArgAsnIleGlyTyr     230235240     TTTATCCTGCAAACATACATGCCTTCCATCCTGATTACCATCCTCTCC1004     PheIleLeuGlnThrTyrMetProSerIleLeuIleThrIleLeuSer     245250255260     TGGGTCTCCTTCTGGATTAATTACGATGCTTCAGCTGCAAGGGTGGCA1052     TrpValSerPheTrpIleAsnTyrAspAlaSerAlaAlaArgValAla     265270275     TTAGGAATCACAACTGTCCTCACAATGACCACAATCAACACCCACCTC1100     LeuGlyIleThrThrValLeuThrMetThrThrIleAsnThrHisLeu     280285290     CGGGAAACTCTCCCTAAAATCCCCTATGTGAAGGCCATTGACATGTAC1148     ArgGluThrLeuProLysIleProTyrValLysAlaIleAspMetTyr     295300305     CTGATGGGGTGCTTTGTCTTCGTTTTCATGGCCCTTCTGGAATATGCC1196     LeuMetGlyCysPheValPheValPheMetAlaLeuLeuGluTyrAla     310315320     CTAGTCAACTACATCTTCTTTGGGAGGGGGCCCCAACGCCAAAAGAAA1244     LeuValAsnTyrIlePhePheGlyArgGlyProGlnArgGlnLysLys     325330335340     GCAGCTGAGAAGGCTGCCAGTGCCAACAATGAGAAGATGCGCCTGGAT1292     AlaAlaGluLysAlaAlaSerAlaAsnAsnGluLysMetArgLeuAsp     345350355     GTCAACAAGATGGACCCCCATGAGAACATCTTACTGAGCACTCTCGAG1340     ValAsnLysMetAspProHisGluAsnIleLeuLeuSerThrLeuGlu     360365370     ATAAAAAATGAAATGGCCACATCTGAGGCTGTGATGGGACTTGGAGAC1388     IleLysAsnGluMetAlaThrSerGluAlaValMetGlyLeuGlyAsp     375380385     CCCAGAAGCACAATGCTAGCCTATGATGCCTCCAGCATCCAGTATCGG1436     ProArgSerThrMetLeuAlaTyrAspAlaSerSerIleGlnTyrArg     390395400     AAAGCTGGGTTGCCCAGGCATAGTTTTGGCCGAAATGCTCTGGAACGA1484     LysAlaGlyLeuProArgHisSerPheGlyArgAsnAlaLeuGluArg     405410415420     CATGTGGCGCAAAAGAAAAGTCGCCTGAGGAGACGCGCCTCCCAACTG1532     HisValAlaGlnLysLysSerArgLeuArgArgArgAlaSerGlnLeu     425430435     AAAATCACCATCCCTGACTTGACTGATGTGAATGCCATAGATCGGTGG1580     LysIleThrIleProAspLeuThrAspValAsnAlaIleAspArgTrp     440445450     TCCCGCATATTCTTCCCAGTGGTTTTTTCCTTCTTCAACATCGTCTAT1628     SerArgIlePhePheProValValPheSerPhePheAsnIleValTyr     455460465     TGGCTTTATTATGTGAACTAAAACATGGCCTCCCACTGGAAGCAAGGA1676     TrpLeuTyrTyrValAsn     470     CTAGATTCCTCCTCAAACCAGTTGTACAGCCTGATGTAGGACTTGGAAAACACATCAATC1736     CAGGACAAAAGTGACGCTAAAATACCTTAGTTGCTGGCCTATCCTGTGGTCCATTTCATA1796     CCATTTGGGTTGCTTCTGCTAAGTAATGAATACACTAAGGTCCTTGTGGTTTTCCAGTTA1856     AAACGCAAGT1866     (2) INFORMATION FOR SEQ ID NO: 6:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 474 amino acids     (B) TYPE: amino acid     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: protein     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:     MetTrpArgValArgLysArgGlyTyrPheGlyIleTrpSerPhePro     151015     LeuIleIleAlaAlaValCysAlaGlnSerValAsnAspProSerAsn     202530     MetSerLeuValLysGluThrValAspArgLeuLeuLysGlyTyrAsp     354045     IleArgLeuArgProAspPheGlyGlyProProValAlaValGlyMet     505560     AsnIleAspIleAlaSerIleAspMetValSerGluValAsnMetAsp     65707580     TyrThrLeuThrMetTyrPheGlnGlnAlaTrpArgAspLysArgLeu     859095     SerTyrAsnValIleProLeuAsnLeuThrLeuAspAsnArgValAla     100105110     AspGlnLeuTrpValProAspThrTyrPheLeuAsnAspLysLysSer     115120125     PheValHisGlyValThrValLysAsnArgMetIleArgLeuHisPro     130135140     AspGlyThrValLeuTyrGlyLeuArgIleThrThrThrAlaAlaCys     145150155160     MetMetAspLeuArgArgTyrProLeuAspGluGlnAsnCysThrLeu     165170175     GluIleGluSerTyrGlyTyrThrThrAspAspIleGluPheTyrTrp     180185190     ArgGlyAspAspAsnAlaValThrGlyValThrLysIleGluLeuPro     195200205     GlnPheSerIleValAspTyrLysLeuIleThrLysLysValValPhe     210215220     SerThrGlySerTyrProArgLeuSerLeuSerPheLysLeuLysArg     225230235240     AsnIleGlyTyrPheIleLeuGlnThrTyrMetProSerIleLeuIle     245250255     ThrIleLeuSerTrpValSerPheTrpIleAsnTyrAspAlaSerAla     260265270     AlaArgValAlaLeuGlyIleThrThrValLeuThrMetThrThrIle     275280285     AsnThrHisLeuArgGluThrLeuProLysIleProTyrValLysAla     290295300     IleAspMetTyrLeuMetGlyCysPheValPheValPheMetAlaLeu     305310315320     LeuGluTyrAlaLeuValAsnTyrIlePhePheGlyArgGlyProGln     325330335     ArgGlnLysLysAlaAlaGluLysAlaAlaSerAlaAsnAsnGluLys     340345350     MetArgLeuAspValAsnLysMetAspProHisGluAsnIleLeuLeu     355360365     SerThrLeuGluIleLysAsnGluMetAlaThrSerGluAlaValMet     370375380     GlyLeuGlyAspProArgSerThrMetLeuAlaTyrAspAlaSerSer     385390395400     IleGlnTyrArgLysAlaGlyLeuProArgHisSerPheGlyArgAsn     405410415     AlaLeuGluArgHisValAlaGlnLysLysSerArgLeuArgArgArg     420425430     AlaSerGlnLeuLysIleThrIleProAspLeuThrAspValAsnAla     435440445     IleAspArgTrpSerArgIlePhePheProValValPheSerPhePhe     450455460     AsnIleValTyrTrpLeuTyrTyrValAsn     465470     (2) INFORMATION FOR SEQ ID NO: 7:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 2189 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: cDNA     (ix) FEATURE:     (A) NAME/KEY: CDS     (B) LOCATION: 214..1566     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:     CCTAGCGCTCCTCTCCGGCTTCCACCAGCCCATCGCTCCACGCTCTCTTGGCTGCTGCAG60     TCTCGGTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTC120     TCTCTCTCTCTCTCTCCCAAGTTTCCTATCTCGTCAAGATCAGGGCAAAAGAAGAAAACA180     CCGAATTCTGCTTGCCGTTTCAGAGCGGCGGTGATGAAGACAAAATTGAACATC234     MetLysThrLysLeuAsnIle     15     TACAACATCGAGTTCCTGCTTTTTGTTTTCTTGGTGTGGGACCCTGCC282     TyrAsnIleGluPheLeuLeuPheValPheLeuValTrpAspProAla     101520     AGGTTGGTGCTGGCTAACATCCAAGAAGATGAGGCTAAAAATAACATT330     ArgLeuValLeuAlaAsnIleGlnGluAspGluAlaLysAsnAsnIle     253035     ACCATCTTTACGAGAATTCTTGACAGACTTCTGGATGGTTACGATAAT378     ThrIlePheThrArgIleLeuAspArgLeuLeuAspGlyTyrAspAsn     40455055     CGGCTTAGACCAGGACTGGGAGACAGTATTACTGAAGTCTTCACTAAC426     ArgLeuArgProGlyLeuGlyAspSerIleThrGluValPheThrAsn     606570     ATCTACGTGACCAGTTTTGGCCCTGTCTCAGATACAGATATGGAATAT474     IleTyrValThrSerPheGlyProValSerAspThrAspMetGluTyr     758085     ACAATTGATGTTTTCTTTCGACAAAAATGGAAAGATGAACGTTTAAAA522     ThrIleAspValPhePheArgGlnLysTrpLysAspGluArgLeuLys     9095100     TTTAAAGGTCCTATGAATATCCTTCGACTAAACAATTTAATGGCTAGC570     PheLysGlyProMetAsnIleLeuArgLeuAsnAsnLeuMetAlaSer     105110115     AAAATCTGGACTCCAGATACCTTTTTTCACAATGGGAAGAAATCAGTA618     LysIleTrpThrProAspThrPhePheHisAsnGlyLysLysSerVal     120125130135     GCTCATAATATGACAATGCCAAATAAGTTGCTTCGAATTCAGGATGAT666     AlaHisAsnMetThrMetProAsnLysLeuLeuArgIleGlnAspAsp     140145150     GGGACTCTGCTGTATACCATGAGGCTTACAGTTCAAGCTGAATGCCCA714     GlyThrLeuLeuTyrThrMetArgLeuThrValGlnAlaGluCysPro     155160165     ATGCACTTGGAGGATTTCCCAATGGATGCTCATTCATGTCCTCTGAAA762     MetHisLeuGluAspPheProMetAspAlaHisSerCysProLeuLys     170175180     TTTGGCAGCTATGCATATACAACTTCAGAGGTCACTTATATTTGGACT810     PheGlySerTyrAlaTyrThrThrSerGluValThrTyrIleTrpThr     185190195     TACAATGCATCTGATTCAGTACAGGTTGCTCCTGATGGCTCTAGGTTA858     TyrAsnAlaSerAspSerValGlnValAlaProAspGlySerArgLeu     200205210215     AATCAATATGACCTGCTGGGCCAATCAATCGGAAAGGAGACAATTAAA906     AsnGlnTyrAspLeuLeuGlyGlnSerIleGlyLysGluThrIleLys     220225230     TCCAGTACAGGTGAATATACTGTAATGACAGCTCATTTCCACCTGAAA954     SerSerThrGlyGluTyrThrValMetThrAlaHisPheHisLeuLys     235240245     AGAAAAATTGGGTATTTTGTGATTCAAACCTATCTGCCTTGCATCATG1002     ArgLysIleGlyTyrPheValIleGlnThrTyrLeuProCysIleMet     250255260     ACTGTCATTCTCTCCCAAGTTTCATTCTGGCTTAACAGAGAATCTGTG1050     ThrValIleLeuSerGlnValSerPheTrpLeuAsnArgGluSerVal     265270275     CCTGCAAGAACTGTGTTTGGAGTAACAACTGTCCTAACAATGACAACT1098     ProAlaArgThrValPheGlyValThrThrValLeuThrMetThrThr     280285290295     CTAAGCATCAGTGCTCGGAATTCTCTCCCCAAAGTGGCTTATGCAACT1146     LeuSerIleSerAlaArgAsnSerLeuProLysValAlaTyrAlaThr     300305310     GCCATGGACTGGTTTATTGCTGTTTGTTATGCATTTGTGTTCTCTGCC1194     AlaMetAspTrpPheIleAlaValCysTyrAlaPheValPheSerAla     315320325     CTAATTGAATTTGCAACTGTTAATTACTTCACCAAAAGAGGATGGACT1242     LeuIleGluPheAlaThrValAsnTyrPheThrLysArgGlyTrpThr     330335340     TGGGATGGGAAGAGTGTAGTAAATGACAAGAAAAAAGAAAAGGCTTCC1290     TrpAspGlyLysSerValValAsnAspLysLysLysGluLysAlaSer     345350355     GTTATGATACAGAACAACGCTTATGCAGTGGCTGTTGCCAATTATGCC1338     ValMetIleGlnAsnAsnAlaTyrAlaValAlaValAlaAsnTyrAla     360365370375     CCGAATCTTTCAAAAGATCCAGTTCTCTCCACCATCTCCAAGAGTGCA1386     ProAsnLeuSerLysAspProValLeuSerThrIleSerLysSerAla     380385390     ACCACGCCAGAACCCAACAAGAAGCCAGAAAACAAGCCAGCTGAAGCA1434     ThrThrProGluProAsnLysLysProGluAsnLysProAlaGluAla     395400405     AAGAAAACTTTCAACAGTGTTAGCAAAATTGACAGAATGTCCAGAATA1482     LysLysThrPheAsnSerValSerLysIleAspArgMetSerArgIle     410415420     GTTTTTCCAGTTTTGTTTGGTACCTTTAATTTAGTTTACTGGGCTACA1530     ValPheProValLeuPheGlyThrPheAsnLeuValTyrTrpAlaThr     425430435     TATTTAAACAGAGAACCTGTATTAGGGGTCAGTCCTTGAATTGAGA1576     TyrLeuAsnArgGluProValLeuGlyValSerPro     440445450     CCCATGTTATCTTTGGGATGTATAGCAACATTAAATTTGGTTTGTTTTGCTATGTACAGT1636     CTGACTAATAACTGCTAATTTGTGATCCAACATGTACAGTATGTATATAGTGACATAGCT1696     TACCAGTAGACCTTTAATGGAGACATGCATTTGCTAACTCATGGAACTGCAGACAGAAAG1756     CACTCCATGCGAAAACAGCCATTGCCTTTTTTAAAGATTTACCCTAGGACCTGATTTAAA1816     GTGAATTTCAAGTGACCTGATTAATTTCCTATTCTTCCAAATGAGATGAAAATGGGGATC1876     CTGTACAACCCTTTGTGGACCCTTTTGGTTTAGCTCTTAAGTAGGGGTATTTTCTACTGT1936     TGCTTAATTATGATGGAAGATAACATTGTCATTCCTAGATGAATCCTTTGAAGTAACAAA1996     CATTGTATCTGACATCAGCTCTGTTCATGAGTGCTCAGAGTCCCTGCTAATGTAATTGGA2056     AGCTTGGTACACATAAGAAAAACTAGAGATTTGAAATCTAGCTATGAATTACTCTATATA2116     GTATCTATAGCCATGTACATATTACAGCATGACAAGCTCGAAATAATTATGAGTCAGCCC2176     GAAAGATGTTAAT2189     (2) INFORMATION FOR SEQ ID NO: 8:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 451 amino acids     (B) TYPE: amino acid     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: protein     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:     MetLysThrLysLeuAsnIleTyrAsnIleGluPheLeuLeuPheVal     151015     PheLeuValTrpAspProAlaArgLeuValLeuAlaAsnIleGlnGlu     202530     AspGluAlaLysAsnAsnIleThrIlePheThrArgIleLeuAspArg     354045     LeuLeuAspGlyTyrAspAsnArgLeuArgProGlyLeuGlyAspSer     505560     IleThrGluValPheThrAsnIleTyrValThrSerPheGlyProVal     65707580     SerAspThrAspMetGluTyrThrIleAspValPhePheArgGlnLys     859095     TrpLysAspGluArgLeuLysPheLysGlyProMetAsnIleLeuArg     100105110     LeuAsnAsnLeuMetAlaSerLysIleTrpThrProAspThrPhePhe     115120125     HisAsnGlyLysLysSerValAlaHisAsnMetThrMetProAsnLys     130135140     LeuLeuArgIleGlnAspAspGlyThrLeuLeuTyrThrMetArgLeu     145150155160     ThrValGlnAlaGluCysProMetHisLeuGluAspPheProMetAsp     165170175     AlaHisSerCysProLeuLysPheGlySerTyrAlaTyrThrThrSer     180185190     GluValThrTyrIleTrpThrTyrAsnAlaSerAspSerValGlnVal     195200205     AlaProAspGlySerArgLeuAsnGlnTyrAspLeuLeuGlyGlnSer     210215220     IleGlyLysGluThrIleLysSerSerThrGlyGluTyrThrValMet     225230235240     ThrAlaHisPheHisLeuLysArgLysIleGlyTyrPheValIleGln     245250255     ThrTyrLeuProCysIleMetThrValIleLeuSerGlnValSerPhe     260265270     TrpLeuAsnArgGluSerValProAlaArgThrValPheGlyValThr     275280285     ThrValLeuThrMetThrThrLeuSerIleSerAlaArgAsnSerLeu     290295300     ProLysValAlaTyrAlaThrAlaMetAspTrpPheIleAlaValCys     305310315320     TyrAlaPheValPheSerAlaLeuIleGluPheAlaThrValAsnTyr     325330335     PheThrLysArgGlyTrpThrTrpAspGlyLysSerValValAsnAsp     340345350     LysLysLysGluLysAlaSerValMetIleGlnAsnAsnAlaTyrAla     355360365     ValAlaValAlaAsnTyrAlaProAsnLeuSerLysAspProValLeu     370375380     SerThrIleSerLysSerAlaThrThrProGluProAsnLysLysPro     385390395400     GluAsnLysProAlaGluAlaLysLysThrPheAsnSerValSerLys     405410415     IleAspArgMetSerArgIleValPheProValLeuPheGlyThrPhe     420425430     AsnLeuValTyrTrpAlaThrTyrLeuAsnArgGluProValLeuGly     435440445     ValSerPro     450     (2) INFORMATION FOR SEQ ID NO: 9:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 1638 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: cDNA     (ix) FEATURE:     (A) NAME/KEY: CDS     (B) LOCATION: 87..1562     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:     GAATTCCCTTGTTTCAGTTCATTCATCCTTCTCTCCTTTCCGCTCAGACTGTAGAGCTCG60     GTCTCTCCAAGTTTGTGCCTAAGAAGATGATAATCACACAAACAAGTCACTGT113     MetIleIleThrGlnThrSerHisCys     15     TACATGACCAGCCTTGGGATTCTTTTCCTGATTAATATTCTCCCTGGA161     TyrMetThrSerLeuGlyIleLeuPheLeuIleAsnIleLeuProGly     10152025     ACCACTGGTCAAGGGGAATCAAGACGACAAGAACCCGGGGACTTTGTG209     ThrThrGlyGlnGlyGluSerArgArgGlnGluProGlyAspPheVal     303540     AAGCAGGACATTGGCGGGCTGTCTCCTAAGCATGCCCCAGATATTCCT257     LysGlnAspIleGlyGlyLeuSerProLysHisAlaProAspIlePro     455055     GATGACAGCACTGACAACATCACTATCTTCACCAGAATCTTGGATCGT305     AspAspSerThrAspAsnIleThrIlePheThrArgIleLeuAspArg     606570     CTTCTGGACGGCTATGACAACCGGCTGCGACCTGGGCTTGGAGATGCA353     LeuLeuAspGlyTyrAspAsnArgLeuArgProGlyLeuGlyAspAla     758085     GTGACTGAAGTGAAGACTGACATCTACGTGACCAGTTTTGGCCCTGTG401     ValThrGluValLysThrAspIleTyrValThrSerPheGlyProVal     9095100105     TCAGACACTGACATGGAGTACACTATTGATGTATTTTTTCGGCAGACA449     SerAspThrAspMetGluTyrThrIleAspValPhePheArgGlnThr     110115120     TGGCATGATGAAAGACTGAAATTTGATGGCCCCATGAAGATCCTTCCA497     TrpHisAspGluArgLeuLysPheAspGlyProMetLysIleLeuPro     125130135     CTGAACAATCTCCTGGCTAGTAAGATCTGGACACCGGACACCTTCTTC545     LeuAsnAsnLeuLeuAlaSerLysIleTrpThrProAspThrPhePhe     140145150     CACAATGGCAAGAAATCAGTGGCTCATAACATGACCACGCCCAACAAG593     HisAsnGlyLysLysSerValAlaHisAsnMetThrThrProAsnLys     155160165     CTGCTCAGATTGGTGGACAACGGAACCCTCCTCTATACAATGAGGTTA641     LeuLeuArgLeuValAspAsnGlyThrLeuLeuTyrThrMetArgLeu     170175180185     ACAATTCATGCTGAGTGTCCCATGCATTTGGAAGATTTTCCCATGGAT689     ThrIleHisAlaGluCysProMetHisLeuGluAspPheProMetAsp     190195200     GTGCATGCCTGCCCACTGAAGTTTGGAAGCTATGCCTATACAACAGCT737     ValHisAlaCysProLeuLysPheGlySerTyrAlaTyrThrThrAla     205210215     GAAGTGGTTTATTCTTGGACTCTCGGAAAGAACAAATCCGTGGAAGTG785     GluValValTyrSerTrpThrLeuGlyLysAsnLysSerValGluVal     220225230     GCACAGGATGGTTCTCGCTTGAACCAGTATGACCTTTTGGGCCATGTT833     AlaGlnAspGlySerArgLeuAsnGlnTyrAspLeuLeuGlyHisVal     235240245     GTTGGGACAGAGATAATCCGGTCTAGTACAGGAGAATATGTCGTCATG881     ValGlyThrGluIleIleArgSerSerThrGlyGluTyrValValMet     250255260265     ACAACCCACTTCCATCTCAAGCGAAAAATTGGCTACTTTGTGATCCAG929     ThrThrHisPheHisLeuLysArgLysIleGlyTyrPheValIleGln     270275280     ACCTACTTGCCATGTATCATGACTGTCATTCTGTCACAAGTGTCGTTC977     ThrTyrLeuProCysIleMetThrValIleLeuSerGlnValSerPhe     285290295     TGGCTCAACAGAGAGTCTGTTCCTGCCCGTACAGTCTTTGGTGTCACC1025     TrpLeuAsnArgGluSerValProAlaArgThrValPheGlyValThr     300305310     ACTGTGCTTACCATGACCACCTTGAGTATCAGTGCCAGAAATTCCTTA1073     ThrValLeuThrMetThrThrLeuSerIleSerAlaArgAsnSerLeu     315320325     CCTAAAGTGGCATATGCGACGGCCATGGACTGGTTCATAGCCGTCTGT1121     ProLysValAlaTyrAlaThrAlaMetAspTrpPheIleAlaValCys     330335340345     TATGCCTTTGTATTTTCTGCACTGATTGAATTTGCCACTGTCAACTAT1169     TyrAlaPheValPheSerAlaLeuIleGluPheAlaThrValAsnTyr     350355360     TTCACCAAGCGGAGTTGGGCTTGGGAAGGCAAGAAGGTGCCAGAGGCC1217     PheThrLysArgSerTrpAlaTrpGluGlyLysLysValProGluAla     365370375     CTGGAGATGAAGAAGAAAACACCAGCAGCCCCAGCAAAGAAAACCAGC1265     LeuGluMetLysLysLysThrProAlaAlaProAlaLysLysThrSer     380385390     ACTACCTTCAACATCGTGGGGACCACCTATCCCATCAACCTGGCCAAG1313     ThrThrPheAsnIleValGlyThrThrTyrProIleAsnLeuAlaLys     395400405     GACACTGAATTTTCCACCATCTCCAAGGGCGCTGCTCCCAGTGCCTCC1361     AspThrGluPheSerThrIleSerLysGlyAlaAlaProSerAlaSer     410415420425     TCAACCCCAACAATCATTGCTTCACCCAAGGCCACCTACGTGCAGGAC1409     SerThrProThrIleIleAlaSerProLysAlaThrTyrValGlnAsp     430435440     AGCCCGACTGAGACCAAGACCTACAACAGTGTCAGCAAGGTTGACAAA1457     SerProThrGluThrLysThrTyrAsnSerValSerLysValAspLys     445450455     ATTTCCCGCATCATCTTTCCTGTGCTCTTTGCCATATTCAATCTGGTC1505     IleSerArgIleIlePheProValLeuPheAlaIlePheAsnLeuVal     460465470     TATTGGGCCACATATGTCAACCGGGAGTCAGCTATCAAGGGCATGATC1553     TyrTrpAlaThrTyrValAsnArgGluSerAlaIleLysGlyMetIle     475480485     CGCAAACAGTAGATAGTGGCAGTGCAGCAACCAGAGCACTGTATACCCC1602     ArgLysGln     490     GTGAAGCATCCAGGCACCCAAACCCCGGGGCTCCCC1638     (2) INFORMATION FOR SEQ ID NO: 10:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 492 amino acids     (B) TYPE: amino acid     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: protein     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:     MetIleIleThrGlnThrSerHisCysTyrMetThrSerLeuGlyIle     151015     LeuPheLeuIleAsnIleLeuProGlyThrThrGlyGlnGlyGluSer     202530     ArgArgGlnGluProGlyAspPheValLysGlnAspIleGlyGlyLeu     354045     SerProLysHisAlaProAspIleProAspAspSerThrAspAsnIle     505560     ThrIlePheThrArgIleLeuAspArgLeuLeuAspGlyTyrAspAsn     65707580     ArgLeuArgProGlyLeuGlyAspAlaValThrGluValLysThrAsp     859095     IleTyrValThrSerPheGlyProValSerAspThrAspMetGluTyr     100105110     ThrIleAspValPhePheArgGlnThrTrpHisAspGluArgLeuLys     115120125     PheAspGlyProMetLysIleLeuProLeuAsnAsnLeuLeuAlaSer     130135140     LysIleTrpThrProAspThrPhePheHisAsnGlyLysLysSerVal     145150155160     AlaHisAsnMetThrThrProAsnLysLeuLeuArgLeuValAspAsn     165170175     GlyThrLeuLeuTyrThrMetArgLeuThrIleHisAlaGluCysPro     180185190     MetHisLeuGluAspPheProMetAspValHisAlaCysProLeuLys     195200205     PheGlySerTyrAlaTyrThrThrAlaGluValValTyrSerTrpThr     210215220     LeuGlyLysAsnLysSerValGluValAlaGlnAspGlySerArgLeu     225230235240     AsnGlnTyrAspLeuLeuGlyHisValValGlyThrGluIleIleArg     245250255     SerSerThrGlyGluTyrValValMetThrThrHisPheHisLeuLys     260265270     ArgLysIleGlyTyrPheValIleGlnThrTyrLeuProCysIleMet     275280285     ThrValIleLeuSerGlnValSerPheTrpLeuAsnArgGluSerVal     290295300     ProAlaArgThrValPheGlyValThrThrValLeuThrMetThrThr     305310315320     LeuSerIleSerAlaArgAsnSerLeuProLysValAlaTyrAlaThr     325330335     AlaMetAspTrpPheIleAlaValCysTyrAlaPheValPheSerAla     340345350     LeuIleGluPheAlaThrValAsnTyrPheThrLysArgSerTrpAla     355360365     TrpGluGlyLysLysValProGluAlaLeuGluMetLysLysLysThr     370375380     ProAlaAlaProAlaLysLysThrSerThrThrPheAsnIleValGly     385390395400     ThrThrTyrProIleAsnLeuAlaLysAspThrGluPheSerThrIle     405410415     SerLysGlyAlaAlaProSerAlaSerSerThrProThrIleIleAla     420425430     SerProLysAlaThrTyrValGlnAspSerProThrGluThrLysThr     435440445     TyrAsnSerValSerLysValAspLysIleSerArgIleIlePhePro     450455460     ValLeuPheAlaIlePheAsnLeuValTyrTrpAlaThrTyrValAsn     465470475480     ArgGluSerAlaIleLysGlyMetIleArgLysGln     485490     (2) INFORMATION FOR SEQ ID NO: 11:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 32 nucleotides     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: cDNA     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:     AGCGCGCGTAATACGACTCACTATAGGGCGAA32     (2) INFORMATION FOR SEQ ID NO: 12:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 26 nucleotides     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: cDNA     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:     CAGCATGAATTGTTAACCTCATTGTA26     (2) INFORMATION FOR SEQ ID NO: 13:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 34 nucleotides     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: cDNA     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:     ATTATTCAAGCTTGCCATGGACAATGGAATGCTC34     (2) INFORMATION FOR SEQ ID NO: 14:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 28 nucleotides     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: cDNA     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:     GGTTTCCAGCTTACTTTGGAGAGGTAGC28     (2) INFORMATION FOR SEQ ID NO: 15:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 25 nucleotides     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: cDNA     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:     GAGGAAGAATTCAGGAGGGTGACCT25     (2) INFORMATION FOR SEQ ID NO: 16:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 29 nucleotides     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: cDNA     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:     GAAAATAACGAATTCCAGTGTCCAGCTTT29     (2) INFORMATION FOR SEQ ID NO: 17:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 32 nucleotides     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: cDNA     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:     CAAAAGAATTCAGCTGAGAAAGCTGCTAATGC32     (2) INFORMATION FOR SEQ ID NO: 18:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 34 nucleotides     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: cDNA     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:     TCAGGCGAATTCTCTTTTGTGCCACATGTCGTTC34     (2) INFORMATION FOR SEQ ID NO: 19:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 32 nucleotides     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: cDNA     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:     AGCGCGCGTAATACGACTCACTATAGGGCGAA32     (2) INFORMATION FOR SEQ ID NO: 20:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 24 nucleotides     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: cDNA     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:     CATCCAGTGGGTACCTCCTTAGGT24     (2) INFORMATION FOR SEQ ID NO: 21:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 23 nucleotides     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: cDNA     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:     GCAATGAAAATCCGGACTGGCAT23     (2) INFORMATION FOR SEQ ID NO: 22:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 28 nucleotides     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: cDNA     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:     GGGAGGGAAGCTTCTGCAACCAAGAGGC28     (2) INFORMATION FOR SEQ ID NO: 23:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 27 nucleotides     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: cDNA     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23:     ACCACATAGAAGCTTATTTAAGTGGAC27     (2) INFORMATION FOR SEQ ID NO: 24:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 33 nucleotides     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: cDNA     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:     TAATCAAGCTTAGTAATGTGGACAGTACAAAAT33     (2) INFORMATION FOR SEQ ID NO: 25:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 33 nucleotides     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: cDNA     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25:     AAATGGAAGCTTTAGAACAGACCTCAGTGTACA33     __________________________________________________________________________ 

We claim:
 1. A rodent fibroblast cell line stably co-transfected with DNA expressing a human GABA_(A) receptor comprising the α₁ β₃ γ₂ subunit combination.
 2. A cell line of claim 1 which is a mouse fibroblast Ltk⁻ cell line.
 3. A rodent fibroblast cell line stably co-transfected with DNA expressing a human GABA_(A) receptor comprising the α₂ β₃ γ₂ subunit combination.
 4. A cell line of claim 3 which is a mouse fibroblast Ltk⁻ cell line.
 5. A rodent fibroblast cell line stably co-transfected with DNA expressing a human GABA_(A) receptor comprising the α₅ β₃ γ₂ subunit combination.
 6. A cell line of claim 5 which is a mouse fibroblast Ltk⁻ cell line.
 7. A rodent fibroblast cell line stably co-transfected with DNA expressing a human GABA_(A) receptor comprising the α₁ β₁ γ_(2S) subunit combination.
 8. A cell line of claim 7 which is a mouse fibroblast Ltk⁻ cell line.
 9. A rodent fibroblast cell line stably co-transfected with DNA expressing a human GABA_(A) receptor comprising the α₁ β₂ γ₂ subunit combination.
 10. A cell line of claim 9 which is a mouse fibroblast Ltk⁻ cell line.
 11. A rodent fibroblast cell line stably co-transfected with DNA expressing a human GABA_(A) receptor comprising the α₃ β₃ γ₂ subunit combination.
 12. A cell line of claim 11 which is a mouse fibroblast Ltk⁻ cell line.
 13. A rodent fibroblast cell line stably co-transfected with DNA expressing a human GABA_(A) receptor comprising the α₆ β₃ γ₂ subunit combination.
 14. A cell line of claim 13 which is a mouse fibroblast Ltk⁻ cell line.
 15. A membrane preparation derived from a rodent fibroblast cell line stably co-transfected with DNA expressing a human GABA_(A) receptor wherein said human GABA_(A) receptor is comprised of a subunit combination selected from a group consisting of α₁ β₃ δγ₂, α₂ β₃ δγ₂, and α₅ β₃ δγ₂.
 16. A preparation as claimed in claim 15 containing a human GABA_(A) receptor consisting of the α₁ β₃ γ_(2S), α₂ β₃ γ_(2S) or α₅ β₃ γ_(2S) subunit combination isolated from stably co-transfected mouse Ltk⁻ fibroblast cells.
 17. The membrane preparation of claim 15 wherein said rodent fibroblast cell line is a mouse fibroblast Ltk⁻ cell line.
 18. A membrane preparation derived from a rodent fibroblast cell line stably co-transfected with DNA expressing a human GABA_(A) receptor wherein said human GABA_(A) receptor is comprised of a subunit combination selected from a group consisting of α₁ β₁ γ_(2S), α₁ β₂ γ₂, α₃ β₃ γ₂, and α₆ β₃ γ₂.
 19. A preparation as claimed in claim 18 containing a human GABA_(A) receptor consisting of the α₁ β₁ γ_(2S), α₁ β₂ γ_(2S), α₃ β₃ γ_(2S) or α₆ β₃ γ_(2S) subunit combination isolated from stably co-transfected mouse Ltk⁻ fibroblast cells.
 20. The membrane preparation of claim 18 wherein said rodent fibroblast cell line is a mouse fibroblast Ltk⁻ cell line.
 21. A method of screening for and designing a medicament which interacts with the human GABA_(A) receptor, which comprises:a) expressing a human recombinant GABA_(A) receptor complex within a rodent fibroblast cell line stably co-transfected with DNA expressing said recombinant human GABA_(A) receptor wherein said recombinant human GABA_(A) receptor comprises a subunit combination selected from the group consisting of α₁ β₃ γ₂, α₂ β₃ γ₂, α₅ β₃ γ₂, α₁ β₁ γ_(2S), α₁ β₂ γ₂, α₃ β₃ γ₂, and α₆ β₃ γ₂ ; b) incubating said rodent fibroblast cell line with at least one chemical compound; and, c) measuring the effect of said compound on the biological activity of said recombinant human GABA_(A) receptor.
 22. The method of claim 21 wherein said rodent fibroblast cell line is a mouse fibroblast Ltk⁻ cell line.
 23. A method of screening for and designing a medicament which interacts with the human GABA_(A) receptor, which comprises:a) expressing a human recombinant GABA_(A) receptor complex within a rodent fibroblast cell line stably co-transfected with DNA expressing said recombinant human GABA_(A) receptor wherein said recombinant human GABA_(A) receptor comprises a subunit combination selected from the group consisting of α₁ β₃ γ₂, α₂ β₃ γ₂, α₅ β₃ γ₂, α₁ β₁ γ_(2S), α₁ β₂ γ₂, α₃ β₃ γ₂, and α₆ β₃ γ₂ ; b) preparing a membrane fraction from said rodent fibroblast cell line; c) incubating said rodent fibroblast cell line with at least one chemical compound; and, d) measuring the effect of said compound on the biological activity of said recombinant human GABA_(A) receptor.
 24. The method of claim 23 wherein said rodent fibroblast cell line is a mouse fibroblast Ltk⁻ cell line. 